We will be following the 3-D spatial trajectory taken by individual IgE molecules as they journey through a rat mast cell. The studies will be made on a novel confocal microscope under development in our lab that is capable of following the motion of a single fluorescent molecule in 3 dimensions. We will study the receptor mediated endocytosis of FceRI receptors on the rat mast cell line RBL-2H3. The IgE-FceRI recognition system is a vital part of the human allergic response and a good model system for following the spatial and kinetic details of intricate, multi-component signal transduction cascades. Complex, multi-component signaling pathways such as this are very important not only in mounting an allergic response, but also in the cell division and growth that is stimulated by external growth factors. Signal transduction cascades that have run haywire cause not only hypersensitive allergies and anaphylaxis, but often cancer. The disruption of these pathways is a clear target for therapeutics for treatment. Proper and controlled disruption of these pathways, however, demands a better knowledge-base concerning the exact proteins involved, and their kinetic and spatial relation to one another. The work proposed herein has the potential to add substantively to this knowledge base by following individual IgE movements in 3-D inside a mast cell. In these studies, IgE will be fluorescently labeled with a highly photostable quantum dot and bound to its high affinity receptor on mast cells. Adding a polyvalent antigen will lead to IgE cross linking, starting a phosphorylation cascade. As a means of down-regulating this cascade, some of the IgE receptor complexes are recruited to clathrin coated pits and internalized, where they are transported to a succession of early and late endosomal compartments. We will follow the spatial and temporal dynamics of this internalization in 3 dimensions with single molecule sensitivity. The specific aims of this work include: Aim 1. Experimentally determine the rates of motion we can follow and the spatial accuracy for simple test systems and verify we can follow individual quantum dots in a cell. Aim 2. We hypothesize that there will be distinctly different and classifiable transport modes for IgE along its internalization path that depend on spatial location. This work advances the development of instrumentation to follow the motion of individual fluorescently labeled molecules in three dimensions. This instrument enables one to follow the 3 dimensional position of a selected protein in real time to see where it goes, how long it spends there, how it gets from A to B, and potentially the conformation of the molecule at its current location. This technology will eventually be used to lead to new insights on protein-protein interactions and help better elucidate complex signal transduction cascades, such as those pathways corrupted in certain cancers, the human allergic response, or host cell functions that are "hi-jacked" by invasive bacteria and viruses.